[Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office (2007). The entire text is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.]

Age

embryo

Gender

Male

Strain

129X1/SvJ

Storage Conditions

liquid nitrogen vapor phase

Karyotype

40 XY, diploid

Derivation

The 129Sv/J ES line was derived from a 129Sv/J (129X1Sv/J in the current nomenclature) strain of mouse, allowing for production of knock-out and transgenic mice. 129Sv/J is a parental substrain of 129 strains and has a white-bellied, light chinchilla or albino coat color with pink eyed appearance.

Clinical Data

Male

Comments

This mouse ES cell line has been shown to be germline competent. 129Sv/J is a parental substrain of 129 strains and has a white-bellied, light chinchilla or albino coat color with pink eyed appearance. This strain is homozygous for Cdh23ahl, the age-related hearing loss 1 mutation, resulting in progressive hearing loss with onset prior to 3 months of age (http://jaxmice.jax.org/strain/000691.html).

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.

One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.

Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.

Spin the cells at 270 x g for 5 min. Aspirate the supernatant.

Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm2.

Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.

Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.

Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact Washington University in St. Louis' Office of Technology Management Development at kratochj@wustl.edu or 314-747-0923.